Part:BBa_K1055007

FsC: functional cutinase

The part enables the user to produce functional cutinase from fungus Fusarium solani pisi which is a cuticula degrading hydrolase. The enzyme is fused to mRFP to allow tracking of expression and also provides a His-Tag for subsequent purification. Both, mRFP and His-Tag, can be cleaved using TEV-Protease.

For detailed information please refer to our http://2013.igem.org/Team:TU_Darmstadt/modelling/Structure wiki page.

Applications of BBa_K1055007

This part is an improvement of the last years BioBrick BBa_K808025 to control and observe its expression even better then before.

To achieve this goal, first, the FSC coding sequence has to be amplified out of the pET22b(+):BBa K808025 plasmid (derived from Sven Jager, TU-Darmstadt). This plasmid includes the FSC gene fused to the gene of mRFP1 (Part:BBa_E1010). Due to this, we were able to quantify the production process of the FSC. Moreover, this approach will result in a convenient screening of mutants and wild type enzyme, using IPTG in LB agar plates (e.g 0.1 to 0.5mM). The amplified gene will be in frame with the pelb leader sequence in order to direct the FSC to E.colis' periplasm. This feature is important for the maturation of the three disulfide bonds. These bonds are only formed under oxidative conditions. Without this feature the FSC will be unfunctional. Additionally, we take the ribosom binding site from the pET22b(+) vector. Moreover, to achieve high purity of the protein, the FSC fusion gene will get a His-Tag at the C-terminus. Therefore, we designed overlap primers with the corresponding motive. This feature is important for the purification of the protein of interest. For an easy cleavage of the reporter as well as the His-Tag, we found a TEV site between the N-Terminus of the FSC and the C-terminal region. Furthermore, to optimize the expression and thus receiving a higher protein yield, the FSC (in frame with the pELB leader sequence) will be cloned in front of an arabinose inducible promotor (e.g. pBAD (PID: BBa I0500)). For the backbone we used the overlap fragment from the TLO-CMK inFusion approach.

Figure 1. The final vector construct contains three major parts: the pelB-FsC-mRFP-6xHis construct, the pBAD promotor and the pSB1C3 backbone.

Plus, our dry lab, did a homology model of the hypothetical fusion protein mRFP1-FsC. The result is illustrated below:

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  Illegal NotI site found at 587
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 733
  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 190
  Illegal AgeI site found at 1420
  Illegal AgeI site found at 1532
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 509